Tristetraprolin down-regulates IL-23 expression in colon cancer cells

Interleukin 23 (IL-23) is an inflammatory cytokine that plays an important role in tumor promotion. Expression of IL-23 is increased in cancer cells and correlates with tumor progression. However, the mechanisms regulating IL-23 expression in cancer cells are still unclear. Here we report that tristetraprolin (TTP), an AU-rich element (ARE)-binding protein, inhibits IL-23 production in CT26 mouse colon cancer cells. Overexpression of TTP decreased the stability of IL-23 mRNA and the expression level of IL-23 in CT26 cells. Conversely, inhibition of TTP by siRNA increased IL-23 production. TTP destabilized a luciferase mRNA reporter containing the IL-23 mRNA 3’UTR, which contains five AREs. Analyses of deletion and point mutants of the IL-23 mRNA 3’UTR demonstrated that the ARE cluster between the third and fifth AREs was responsible for TTP-mediated destabilization of IL-23 mRNA. A RNA electrophoretic mobility shift assay confirmed that TTP binds to this ARE cluster. Taken together, these results demonstrate that TTP acts as a negative regulator of IL-23 gene expression in mouse colon cancer cells and suggest its potential application as a novel therapeutic target to control IL-23-mediated tumor promotion.     

CNOT7/hCAF1 is involved in ICAM-1 and IL-8 regulation by tristetraprolin

Tristetraprolin (TTP) is an RNA-binding protein which can bind to the AU-rich elements (AREs) at the 3′-untranslated region (3′-UTR) of target mRNA and promote mRNA deadenylation and degradation. We have shown in a previous study that TTP regulates tumor necrosis factor-α (TNF-α)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8), both of whose mRNAs have AREs in the 3′-UTR, in human pulmonary microvascular endothelial cells (HPMEC) through destabilizing target mRNAs, nevertheless, the mechanism by which TTP promotes mRNA decay remains unclear. Observations have indicated that TTP can interact with CAF1 (CNOT7/hCAF1 in human), a subunit of the CCR4-NOT complex with deadenylase activity. Another study illustrated that TTP can directly bind to CNOT1, the scaffold subunit of the CCR4-NOT complex. The present study showed that TTP bound to the AREs of ICAM-1 and IL-8 mRNAs and was coimmunoprecipitated with intracellular ICAM-1 and IL-8 mRNAs. TTP, CNOT7 and CNOT1 were coimmunoprecipitated in HPMEC. CNOT7 silencing stabilized ICAM-1 and IL-8 mRNAs and increased ICAM-1 and IL-8 production following TNF-α stimulation. These results, together with our previous study, suggest that CNOT7/hCAF1 is involved in ICAM-1 and IL-8 regulation by TTP in HPMEC.     

KSRP suppresses cell invasion and metastasis through miR-23a-mediated EGR3 mRNA degradation in non-small cell lung cancer

KH-type splicing regulatory protein (KSRP) is a single-strand RNA binding protein which regulates mRNA stability either by binding to AU-rich elements (AREs) of mRNA 3′UTR or by facilitating miRNA biogenesis to target mRNA. Unlike its well-characterized function at the molecular level in maintaining RNA homeostasis, the role of KSRP in cancer progression remains largely unknown. Here we investigate the role of KSRP in non-small cell lung cancer (NSCLC). We first examined KSRP expression by immunohistochemistry in a cohort containing 196 NSCLC patients and observed a strong positive correlation between KSRP expression and survival of NSCLC patients. Multivariate analysis further identified KSRP as an independent prognostic factor. Manipulating KSRP expression significantly affected in vitro cell mobility and in vivo metastatic ability of NSCLC cells. Microarray analysis identified an ARE-containing gene, EGR3, as a downstream effector of KSRP in NSCLC. Interestingly, we found that KSRP decreased EGR3 mRNA stability in an ARE-independent manner. By screening KSRP-regulated miRNAs in NSCLC cells, we further found that miR-23a directly binds to EGR3 3′UTR, reducing EGR3 expression and thereby inhibiting NSCLC cell mobility. Our findings implicate a targetable KSRP/miR-23a/EGR3 signaling axis in advanced tumor phenotypes.     

RNA结合蛋白调节mRNA稳定性的作用机制

免疫稳态的维持涉及多种细胞因子的基因表达调控,其中在转录后水平对mRNA稳定性的调控起重要作用。ARE(AU-rich element)位于mRNA 3'UTR(非编码区),富含AU碱基,某些RNA结合蛋白通过识别结合ARE影响mRNA的稳定性。本文综合最新研究,概述了TTP、HUR等RNA结合蛋白对mRNA稳定性的调节机制及其在信号通路中的作用。     

Synthesis of amide and sulfonamide substituted N-aryl 6-aminoquinoxalines as PFKFB3 inhibitors with improved physicochemical properties

In oncology, the “Warburg effect” describes the elevated production of energy by glycolysis in cancer cells. The ubiquitous and hypoxia-induced 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) plays a noteworthy role in the regulation of glycolysis by producing fructose-2,6-biphosphate (F-2,6-BP), a potent activator of the glycolysis rate-limiting phosphofructokinase PFK-1. Series of amides and sulfonamides derivatives based on a N-aryl 6-aminoquinoxaline scaffold were synthesized and tested for their inhibition of PFKFB3 in vitro in a biochemical assay as well as in HCT116 cells. The carboxamide series displayed satisfactory kinetic solubility and metabolic stability, and within this class, potent lead compounds with low nanomolar activity have been identified with a suitable profile for further in vivo evaluation.     

真核mRNA的3′非翻译区转录后水平调控作用研究进展

真核生物mRNA的3′非翻译区(3′_UTR)在基因表达的转录后调控中起着重要作用:3′_UTR内存在末端加工信号以指导mRNA3′末端的加工;3′_UTR不但控制mRNA的稳定性及降解速率、协助辨认特殊密码子,而且还控制着mRNA的翻译时间、位点及控制其翻译起始及效率等。     

Nuclear Targeting of 6-Phosphofructo-2-kinase (PFKFB3) Increases Proliferation via Cyclin-dependent Kinases

The regulation of metabolism and growth must be tightly coupled to guarantee the efficient use of energy and anabolic substrates throughout the cell cycle. Fructose 2,6-bisphosphate (Fru-2,6-BP) is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), a rate-limiting enzyme and essential control point in glycolysis. The concentration of Fru-2,6-BP in mammalian cells is set by four 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1–4), which interconvert fructose 6-phosphate and Fru-2,6-BP. The relative functions of the PFKFB3 and PFKFB4 enzymes are of particular interest because they are activated in human cancers and increased by mitogens and low oxygen. We examined the cellular localization of PFKFB3 and PFKFB4 and unexpectedly found that whereas PFKFB4 localized to the cytoplasm (i.e. the site of glycolysis), PFKFB3 localized to the nucleus. We then overexpressed PFKFB3 and observed no change in glucose metabolism but rather a marked increase in cell proliferation. These effects on proliferation were completely abrogated by mutating either the active site or nuclear localization residues of PFKFB3, demonstrating a requirement for nuclear delivery of Fru-2,6-BP. Using protein array analyses, we then found that ectopic expression of PFKFB3 increased the expression of several key cell cycle proteins, including cyclin-dependent kinase (Cdk)-1, Cdc25C, and cyclin D3 and decreased the expression of the cell cycle inhibitor p27, a universal inhibitor of Cdk-1 and the cell cycle. We also observed that the addition of Fru-2,6-BP to HeLa cell lysates increased the phosphorylation of the Cdk-specific Thr-187 site of p27. Taken together, these observations demonstrate an unexpected role for PFKFB3 in nuclear signaling and indicate that Fru-2,6-BP may couple the activation of glucose metabolism with cell proliferation.Neoplastic transformation and growth require a massive increase in glucose uptake and glycolytic flux not only for energy production but also for the synthesis of nucleic acids, amino acids, and fatty acids. A central control point of glycolysis is the negative allosteric regulation of a rate-limiting enzyme, phosphofructokinase-1 (PFK-1),² by ATP (i.e. the Pasteur effect) (1, 2). When intracellular ATP production exceeds usage, ATP inhibits PFK-1 and glycolytic flux. Fructose 2,6-bisphosphate (Fru-2,6-BP) is a potent allosteric activator of PFK-1 that overrides this inhibitory influence of ATP on PFK-1, allowing forward flux of the entire pathway (3–5).The steady-state cellular concentration of Fru-2,6-BP is dependent on the activities of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB), which are encoded by four independent genes (PFKFB1–4) (6, 7). The PFKFB3 mRNA is distinguished by the presence of multiple copies of an AUUUA instability motif in its 3′-untranslated region and the PFKFB3 protein product has a high kinase:phosphatase activity ratio (740:1) (8). PFKFB3 mRNA is overexpressed by rapidly proliferating transformed cells and the PFKFB3 protein is highly expressed in solid tumors and leukemias (8–11). PFKFB3 expression is increased in response to several mitogenic stimuli, including progesterone, serum, and insulin (12–14). These studies indicate that the PFKFB3 enzyme may serve an essential function in the regulation of glucose metabolism during cell proliferation.The PFKFB3 mRNA is spliced into several variants that encode distinct carboxyl-terminal domains (9, 15). Importantly, the functional consequences of the disparate carboxyl-terminal variants of PFKFB3 are unknown. The mRNA splice variant 5 is the dominant PFKFB3 mRNA in human brain, several transformed cells, and colon adenocarcinoma tissues (9, 10). In the following series of experiments, we present data that the carboxyl-terminal domain of PFKFB3 variant 5 localizes the enzyme to the nucleus where its product, Fru-2,6-BP, increases the expression and activity of cyclin-dependent kinase-1. These data demonstrate a heretofore unidentified function of the PFKFB3 enzyme that is distinct from glycolysis, and provide a potential mechanism for the coupling of metabolism and proliferation.     

MAPK14/p38α-dependent modulation of glucose metabolism affects ROS levels and autophagy during starvation

Increased glycolytic flux is a common feature of many cancer cells, which have adapted their metabolism to maximize glucose incorporation and catabolism to generate ATP and substrates for biosynthetic reactions. Indeed, glycolysis allows a rapid production of ATP and provides metabolic intermediates required for cancer cells growth. Moreover, it makes cancer cells less sensitive to fluctuations of oxygen tension, a condition usually occurring in a newly established tumor environment. Here, we provide evidence for a dual role of MAPK14 in driving a rearrangement of glucose metabolism that contributes to limiting reactive oxygen species (ROS) production and autophagy activation in condition of nutrient deprivation. We demonstrate that MAPK14 is phosphoactivated during nutrient deprivation and affects glucose metabolism at 2 different levels: on the one hand, it increases SLC2A3 mRNA and protein levels, resulting in a higher incorporation of glucose within the cell. This event involves the MAPK14-mediated enhancement of HIF1A protein stability. On the other hand, MAPK14 mediates a metabolic shift from glycolysis to the pentose phosphate pathway (PPP) through the modulation of PFKFB3 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase 3) degradation by the proteasome. This event requires the presence of 2 distinct degradation sequences, KEN box and DSG motif Ser273, which are recognized by 2 different E3 ligase complexes. The mutation of either motif increases PFKFB3 resistance to starvation-induced degradation. The MAPK14-driven metabolic reprogramming sustains the production of NADPH, an important cofactor for many reduction reactions and for the maintenance of the proper intracellular redox environment, resulting in reduced levels of ROS. The final effect is a reduced activation of autophagy and an increased resistance to nutrient deprivation.     

Protein phosphorylation regulates in vitro spinach chloroplast petD mRNA 3′-untranslated region stability,processing, and degradation

RNA-binding proteins (RNPs) participate in diverse processes of mRNA metabolism, and phosphorylation changes their binding properties. In spinach chloroplasts, 24RNP and 28RNP are associated with polynucleotide posphorylase forming a complex on charge of pre-mRNA 3′-end maturation. Here, we tested the hypothesis that the phosphorylation status of 24RNP and 28RNP, present in a spinach chloroplast mRNA 3′-UTR processing extract (CPE), controls the transition between petD precursor stabilization, 3′-UTR processing, and RNA degradation in vitro. The CPE processed or stabilized petD precursor depending on the ATP concentration present in an in vitro 3′-UTR processing (IVP) assay. These effects were also observed when ATP was pre-incubated and removed before the IVP assay. Moreover, a dephosphorylated (DP)-CPE degraded petD precursor and recovered 3′-UTR processing or stabilization activities in an ATP concentration dependent manner. To determine the role 24/28RNP plays in regulating these processes a 24/28RNP-depleted (Δ24/28)CPE was generated. The Δ24/28CPE degraded the petD precursor, but when it was reconstituted with recombinant non-phosphorylated (NP)-24RNP or NP-28RNP, the precursor was stabilized, whereas when Δ24/28CPE was reconstituted with phosphorylated (P)-24RNP or P-28RNP, it recovered 3′-UTR processing, indicating that 24RNP or 28RNP is needed to stabilize the precursor, have a redundant role, and their phosphorylation status regulates the transition between precursor stabilization and 3′-UTR processing. A DP-Δ24/28CPE reconstituted or not with NP-24/28RNP degraded petD precursor. Pre-incubation of DP-Δ24/28CPE with NP-24/28RNP plus 0.03 mM ATP recovered 3′-UTR processing activity, and its reconstitution with P-24/28RNP stabilized the precursor. However, pre-incubation of DP-Δ24/28CPE with 0.03 mM ATP, and further reconstitution with NP-24/28RNP or P-24/28RNP produced precursor stability instead of RNA degradation, and RNA processing instead of precursor stability, respectively. Moreover, in vitro phosphorylation of CPE showed that 24RNP, 28RNP, and other proteins may be phosphorylated. Altogether, these results reveal that phosphorylation of 24RNP, 28RNP, and other unidentified CPE proteins mediates the in vitro interplay between petD precursor stability, 3′-UTR processing, and degradation, and support the idea that protein phosphorylation plays an important role in regulating mRNA metabolism in chloroplast.     

miR-613 inhibits cell migration and invasion by downregulating Daam1 in triple-negative breast cancer

Dishevelled-associated activator of morphogenesis 1 (Daam1) is a formin protein and participates in regulating cell migration of triple-negative breast cancer (TNBC) cells. The specific miRNA targeting Daam1 and mediating cell migration and invasion remains obscure. This experiment investigated the suppressive role of miR-613 in TNBC cells. The luciferase activity of Daam1 3′-untranslated region (3′-UTR) based reporters constructed in HEK-293T and MCF-7 cells suggested that Daam1 was the target gene of miR-613. Overexpressed miR-613 reduced the protein level of Daam1, weakened RhoA activity, and retarded the cell migration, cell invasion and colony formation of TNBC cells. Overexpression of Daam1 or RhoA rescued cell migration and invasion in miR-613-overexpressed TNBC cells, but failed to reverse colony formation. MiR-613 was significantly downregulated in breast cancer tissues compared with that in adjacent normal tissues. This downregulation in TNBC tissues and lymphnode metastatic breast cancer tissues was more obvious than that in non-TNBC tissues and non-metastatic cancer tissues, respectively. MiR-613 weakens the resistance of TNBC cells against paclitaxel rather than adriamycin, cyclophosphamide, docetaxel, and kaempferol. Taken together, miR-613 is involved in cell migration and invasion of TNBC cells via targeting Daam1/RhoA signaling pathway.